and ligand-gated ion channels as common mechan

Environ Sci Pollut Res DOI 10.1007/s11356-013-1759-x

PCB MIXTURES IN A COMPLEX WORLD

Modulation of cell viability, oxidative stress, calcium homeostasis, and voltage- and ligand-gated ion channels as common mechanisms of action of (mixtures of) non-dioxinlike polychlorinated biphenyls and polybrominated diphenyl ethers Remco H. S. Westerink

Received: 2 November 2012 / Accepted: 22 April 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract Non-dioxin-like polychlorinated biphenyls (NDLPCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that exert neurodevelopmental and neurobehavioral effects in vivo in humans and animals. Acute in vitro neurotoxic effects include changes in cell viability, oxidative stress, and basal intracellular calcium levels. Though these acute cellular effects could partly explain the observed in vivo effects, other mechanisms, such as effects on calcium influx and neurotransmitter receptor function, likely contribute to the disturbance in neurotransmission. This concise review combines in vitro data on cell viability, oxidative stress and basal calcium levels with recent data that clearly demonstrate that (hydroxylated) PCBs and (hydroxylated) PBDEs can exert acute effects on voltage-gated Ca2+ channels as well as on excitatory and inhibitory neurotransmitter receptors in vitro. These novel mechanisms of action are shared by NDL-PCBs, OH-PBDEs, and some other persistent organic pollutants, such as tetrabromobisphenol-A, and could have profound effects on neurodevelopment, neurotransmission, and neurobehavior in vivo. Keywords Non-Dioxin-Like Polychlorinated Biphenyls . Polybrominated Diphenyl Ethers . Mixture toxicity . Neurotoxicity . Cell viability . Oxidative stress . Calcium

Responsible editor: Henner Hollert R. H. S. Westerink (*) Neurotoxicology Research Group, Toxicology Division, Institute for Risk Assessment Sciences (IRAS), Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.177, 3508 TD, Utrecht, The Netherlands e-mail: [email protected]

homeostasis . Voltage-gated calcium channels . GABAA receptors . Nicotinic acetylcholine receptors

Introduction Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants (POPs). PCBs and PBDEs have a structural similarity and for both groups of compounds, theoretically 209 different possible congeners exist. PCBs have been used in numerous industrial and commercial applications, whereas PBDEs have been mainly used as flame retardants. Worldwide, more than 1.5 million tons of PCBs were produced until their production, commercialization, and use were largely prohibited in the 1970s and 1980s (Breivik et al. 2002). Despite this ban, PCBs are still present in the environment and in biota, mainly because of improper disposal in combination with their lipophilicity and biopersistence (ATSDR 2000; Consonni et al. 2012). Comparable to PCBs, PBDEs are globally dispersed throughout the environment (reviewed by Law et al. 2008). Since 2003, voluntary phase out of manufacturing PBDEs and use of substitutes have been taking place in North America and Europe, resulting in declining commercial PBDE production. Nonetheless, the levels of some PBDEs in the environment may still be increasing (Darnerud et al. 2001; Hites 2004), with human and environmental levels of PBDEs being approximately one order of magnitude higher in North America than in Europe and Asia (reviewed by Frederiksen et al. 2009). Human exposure to PCBs occurs mainly via food intake (EFSA 2005), although inhalation of (indoor) air and house

Environ Sci Pollut Res

dust also provides significant exposure, mainly to lowerchlorinated congeners (Broding et al. 2007; Hu et al. 2012). Human exposure to PBDEs occurs in particular via air and ingestion of house dust, but also through intake of vegetable and animal products (reviewed by Frederiksen et al. 2009) and in occupational settings (reviewed by Carpenter 2006; Frederiksen et al. 2009). Children are exposed to larger amounts of these POPs than adults (Fischer et al. 2006; Toms et al. 2009), mainly due to their relatively high food intake, child-specific hand-to-mouth behavior, and larger ingestion of house dust. An additional source of exposure for young children is breast milk, in which especially lowerbrominated PBDEs have been detected (reviewed by Frederiksen et al. 2009). Regardless of the route of exposure, levels of individual PCBs and PBDEs in human (cord) blood have been shown to be in the subnanomolar to lower nanomolar range, whereas the sum of the levels of the most common PCBs and PBDEs in blood can add up to the higher nanomolar range, especially in occupationally exposed people (Gabrio et al. 2000; Petrik et al. 2006; reviewed in Dingemans et al. 2011). PCBs are divided into coplanar dioxin-like (DL-) PCBs and non-dioxin-like (NDL-) PCBs. Contrary to DL-PCBs, NDL-PCBs have one or more chlorines in the ortho-positions, resulting in nonplanar structures. As a result, NDLPCBs have little or no affinity to the aryl hydrocarbon receptor (AhR) and display a different toxicological profile (ATSDR 2000; Van den Berg et al. 2006). Epidemiological studies indicated that perinatal exposure to NDL-PCBs can result in neurodevelopmental and neurobehavioral effects in children (for review see Faroon et al. 2001; Winneke et al. 2002; Schantz et al. 2003). More recently, adverse effects on cognitive and neurodevelopmental parameters in humans were also correlated to PBDE exposure. Motor, cognitive, and behavioral performance in 6-year-old Dutch children was correlated with maternal serum levels of PBDEs measured in the 35th week of pregnancy (Roze et al. 2009). In another study, the scores of US children 0–6 years of age on yearly tests of mental and physical development were lower among those prenatally exposed to higher concentrations of PBDEs (Herbstman et al. 2010). These findings are in line with in vivo animal studies that demonstrated that (neonatal) exposure to PCBs and PBDEs can induce a wide range of neurobehavioral effects, including changes in motor activity, spontaneous behavior, learning, memory, and attention (reviewed in Ulbrich and Stahlmann 2004; Costa and Giordano 2007; Fonnum and Mariussen 2009; Dingemans et al. 2011). To date, in vitro studies identified several mechanisms of action for NDL-PCBs and PBDEs, including alterations in cell viability, oxidative stress, calcium homeostasis, as well as on neurotransmitter receptor function (see below), that could underlie these observed in vivo effects in animals and humans.

Effects on cell viability and oxidative stress A reduction in cell viability is a clear, yet not very subtle sign of xenobiotic-induced neurotoxicity. Similarly, xenobiotic-induced increased oxidative stress, e.g., enhanced production of reactive oxygen species (ROS), is a clear indication for neurotoxicity. Both, cell viability and oxidative stress can be assessed in vitro using highthroughput biochemical assays. It is therefore not surprising that many of the early in vitro studies on PCBs, dating back to the 1970s and 1980s, and on PBDEs focused on effects on cell viability. Currently, effects of PCBs and PBDEs on cell viability have been assessed in numerous cell models, including primary cultures, and usually occur in the (tens of) micromolar range. The effects on cell viability often closely correlate with induction of oxidative stress, suggesting that induction of oxidative stress is causally related to the reduction in cell viability (see e.g., Howard et al. 2003; Huang et al. 2010). In support of this, the reduction in cell viability induced by PCBs and PBDEs could be (partially) prevented by the addition of antioxidants such as vitamin E and melatonin (see e.g., Fonnum et al. 2006; Costa and Giordano 2007; Costa et al. 2008; Fonnum and Mariussen 2009; Dingemans et al. 2011 for more extensive reviews on cell viability and oxidative stress). Despite the wealth of available studies and the relative simplicity of the abovementioned endpoints, several issues remain to be completely resolved. These issues are mainly related to bioavailability, bioaccumulation, bioactivation, and the possible occurrence of additive or synergistic effects following exposure to mixtures of POPs. Previous studies indicated that PBDEs can accumulate intracellularly due to their high lipophilicity and, as such, concentrations in the medium may be underestimating the concentration present in the cell by 1-2 orders of magnitude (Mundy et al. 2004; Kodavanti et al. 2005; Huang et al. 2010). On the other hand, inclusion of serum in the exposure medium strongly (∼5-fold) attenuated the intracellular accumulation of PBDEs, indicating reduced bioavailability due to binding of PBDEs to serum proteins (Mundy et al. 2004). Absorption, elimination, and distribution of PCBs and PBDEs have been extensively studied. Importantly, both PCBs and PBDEs can undergo oxidative metabolism in vivo resulting in numerous hydroxylated metabolites at concentrations similar to or sometimes even exceeding those of the unmetabolized parent compounds (see e.g., Costa and Giordano 2007; Fonnum and Mariussen 2009; Dingemans et al. 2011 for more extensive reviews on kinetics). These hydroxylated metabolites are reported to be more potent in reducing cell viability and increasing oxidative stress. The level of oxidative stress in e.g., cerebellar granule cells induced by hydroxylated PCBs is increased ∼10-fold compared to control (Dreiem et al. 2009), whereas parent


compounds were reported to increase ROS levels by less than 2-fold (Mariussen et al. 2002). The degree of bioactivation with respect to cell viability and ROS production is even more pronounced for PBDEs. Hydroxylated PBDEs were shown to more effectively increase cytotoxicity in H295R human adrenocortical carcinoma cells (Cantón et al. 2006), rat PC12 cells (Dingemans et al. 2010a), and chicken DT40 cells (Ji et al. 2011), to increase oxidative stress in human hepatoma HepG2 cells (An et al. 2011) and to increase both cytotoxicity and oxidative stress in embryonic zebrafish (Usenko et al. 2012). Yet, this issue of bioactivation is far from completely resolved as any of the individual PCB or PBDE congeners can give rise to (a mixture of) multiple metabolites yielding an enormous amount of metabolites and possible exposure scenarios. Currently, a direct comparison of the parent compounds and the possible metabolites with respect to cytotoxicity and oxidative stress is often lacking and it is often unclear which of these metabolites can actually be formed in vivo. Another largely unresolved issue is the occurrence of antagonistic, additive, or synergistic effects following exposure to mixtures of environmental pollutants. For DL-PCBs, the toxic and biological effects have been primarily associated with binding and activation of the AhR transduction pathway. With respect to AhR activation, DL-PCBs have been assigned a “toxic equivalent factor” (TEF). The TEF concept relies on the relative effect potency determined for individual polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and PCB congeners for inducing AhR activation relative to the reference compound 2,3,7,8-tetrachlorodibenzo-p-dioxin. When the observed toxic effects of multiple compounds are additive the total toxic equivalent (TEQ, which is defined by the sum of the products of the concentration of each compound multiplied by its TEF value) can be used to estimate the potency of the mixture (Van den Berg et al. 2006). However, despite the fact that induction of cytotoxicity and oxidative stress by PCBs and PBDEs has often been tested using commercial mixtures, such as Aroclor1254 and DE-71, it is still not clearly established whether the TEF concept, and thus additivity, applies for these endpoints. On the other hand, the TEF concept has been proposed for activation of ryanodine (Ry) receptors (RyR) on the endoplasmic reticulum (ER) by NDL-PCBs and PBDEs (Pessah et al. 2010; Kim et al. 2011) and activation of RyR correlated well with cytotoxicity (Kim et al. 2011). Establishing a TEF concept for the different neurotoxic endpoints has been hampered, at least partly, by variability in the composition of commercial mixtures (see e.g., Kodavanti et al. 2001). More importantly, only a few studies directly compare the potency of different individual congeners with the respective mixtures, often yielding contradictory results. In J774A.1 macrophage cells, PCBs 101, 153, and 180 induced a synergistic effect on cell death and apoptosis at concentrations

that were inactive for the individual congeners (Ferrante et al. 2011). Contrary, in human T47D and MDA-MB-231 breast cancer cells, weak antagonism was observed between PCB153 and PCB126 for cytotoxicity and ROS production at lower concentrations (50 % activation (at 10 μM) and >800 % potentiation (at 10 μM), whereas BDE-47 is ineffective (Hendriks et al. 2010). Interestingly, though binary mixtures of NDL-PCBs result in competitive activation and potentiation of human GABAA-Rs, a binary mixture of PCB-47 and 6-OH-PBDE-47 induced an additive activation as well as potentiation of GABAA receptors (Hendriks et al. 2010). Overall, NDL-PCBs and OH-PBDEs thus act as (partial) agonist on the postsynaptic human GABAA-Rs, thereby potentially reducing excitability of the nervous system. Noteworthy in this respect is that e.g. developmental PCB exposure was reported to increase extracellular levels of GABA, thereby potentially contributing to a reduction in neurotransmission (Boix et al. 2010). These findings thus demonstrate that GABAA and nACh receptors are affected differently by PCB-47 and 6-OHPBDE-47, with inhibitory GABAA-mediated signaling being potentiated and excitatory α4β2 nACh-mediated signaling being inhibited. Considering these opposite actions and the additive interaction of the congeners, these effects are likely to be augmented in vivo, resulting in an inhibition of neuronal excitability. This may be further enhanced by additional effects on voltage-gated ion channels, including inhibition of voltage-gated calcium channels by NDL-PCBs and (OH-) BDEs (see above: effects on calcium homeostasis). Moreover, BDE-209 (at ≥0.1 μM) was recently shown to concentration-dependently inhibit voltage-gated sodium channels (VGSCs), shift the activation and inactivation of VGSCs towards hyperpolarizing direction, slow down the recovery from inactivation and decrease the fraction of activated VGSCs (Xing et al. 2010), suggesting that BDE209


could inhibit the generation of action potentials and thus neuronal excitability.

Evidently, NDL-PCBs and (OH-) BDEs can exert neurotoxic effects in vitro, including effects on cell viability, oxidative stress, and basal calcium levels via intracellular calcium stores. Importantly, these effects are nicely correlated, suggesting a causal relation of store-mediated increases in basal [Ca2+]i, increased oxidative stress and caspase activity with reduced cell viability (also see Fig. 1). Of concern is that the store-mediated increases in basal [Ca2+]i, already occur in the low- and even sub-micromolar level. Of similar concern, the acute effects on functional endpoints (calcium homeostasis and neurotransmitter receptor function) also occur already in the low- and even submicromolar level (see Fig. 1). Though some discrepancies exist, the effects on VGCC, VGSC, GABAA receptors, and nACh receptors suggest that (mixtures of) PCBs and PBDEs can reduce neuronal excitability. Consequently, the overall effect in vivo could be even more pronounced, though it should be noted that till date effects of PCBs or PBDEs on many neurotransmitter receptors, e.g., glutamate or glycine receptors, have not or only hardly been studied. On the other hand, the increase in basal [Ca2+]i could not only affect cell viability pathways, but can also increase neuronal excitability, thereby counteracting the suggested reduction in

neuronal excitability. As such, the overall effect on neuronal excitability remains to be determined and potentially differs between in vitro models as well as between different brain areas in vivo due to differences in relative receptor expression. For risk assessment purposes, additional information regarding bioactivation is essential since hydroxylation drastically increases the neurotoxic potency of e.g. BDE-47. Noteworthy is this respect is also the observation that binary mixtures of PCBs with (OH-) BDEs can induce additive effects, whereas additivity is apparently less likely to occur for mixtures consisting of only PCBs or only PBDEs. Hopefully, future research can fully elucidate if and under which conditions additivity applies. Notably, the adverse effects described in this review are not only shared by PCBs and PBDEs, but possibly by other POPs as well. Recently, the brominated flame retardant tetrabromobisphenol-A (TBBPA) has been shown to not only induce cell death and oxidative stress, but also to increase basal [Ca2+]i, inhibit VGCCs and α4β2 nACh-Rs, and to activate/potentiate α1β2γ2 GABAA-Rs (Hendriks et al. 2012b), just as NDL-PCBs and 6-OH-BDE-47. A thorough investigation and comparison of different abundant POPs for these specific endpoints could be a first step towards (mechanistically) investigating the effects of environmentally relevant complex mixtures. Currently, the best studied environmentally relevant combination includes methylmercury (MeHg). Mixtures of PCBs with MeHg have previously been shown to synergistically reduce brain dopamine content in rat striatal slices, while synergistically increasing extracellular

Fig. 1 Schematic representation of the common cellular actions of NDL-PCBs, OH-PBDEs, and some other POPs, such as TBBPA. Neuronal activity can be reduced by inhibitory actions on nicotinic acetylcholine receptors (nACh-R) and voltage-gated calcium channels (VGCC), resulting in reduced neurotransmitter secretion (middle). Moreover, neuronal activity and thus neurotransmitter secretion can be further reduced by inhibitory actions on voltage-gated sodium

channels (VGSC) and activating/potentiating effects on GABAA receptors. In contrast to these inhibitory effects, NDL-PCBs and OHPBDEs can induce release of calcium from intracellular stores, such as the endoplasmic reticulum and mitochondria, which could (temporarily) increase neurotransmitter secretion. More importantly, calciumstore-mediated calcium release could trigger activation of (calciumdependent, caspase-mediated) cell death pathways

Future perspectives


dopamine levels (Bemis and Seegal 1999). Similarly, a mixture of PCB4 with MeHg synergistically increased basal calcium levels in rat cerebellar granule cells, at least when both were applied at low micromolar concentrations (Bemis and Seegal 2000). In line with these findings, co-exposure of MeHg (sub-micromolar range) with PCBs (micromolar range) resulted in additive or slightly synergistic effects on the induction of cell death via activation of calcium-regulated calpains and lysosomal cathepsins, possibly through disruption of mitochondrial function and intracellular calcium signaling in AtT20 pituitary cells (Johansson et al. 2006). Yet, this issue is also far from resolved since more recently coexposure of mouse hippocampal neuronal HT22 cells to MeHg and PCB153 or PCB126 was shown to result mainly in antagonistic effects on cell viability (Tofighi et al. 2011). Until the above matters are investigated more thoroughly, and for different exposure scenarios, risk assessment of mixtures of POPs, including PCBs and PBDEs, will be hampered by a lack of insight into the constrains of additivity and bioactivation with respect to the different neurotoxic endpoints. Acknowledgments Elsa C. Antunes Fernandes, Milou M.L. Dingemans, Hester S. Hendriks, Regina G.D.M. van Kleef, Wendy T. Langeveld, Marieke Meijer, Ad Reniers, and other members of the Neurotoxicology Research Group (IRAS) are gratefully acknowledged for their valuable scientific and technical support throughout the projects that provided the data and insight to prepare this review. I sincerely apologize to all the authors of primary literature or previous reviews that could not be included due to space limitations. This study was supported by the European Union [ATHON: FP7-FOOD-CT-2005-022923; ENFIRO: FP7-ENV-2008-1-226563; DENAMIC: grant no. FP7-ENV-2011282957] and the Faculty of Veterinary Medicine of Utrecht University.

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